Nematodes are small wormlike animals, many of which are plant, animal or human parasites which cause a variety of diseases. Plant pathogenic nematodes are a major agricultural problem causing significant crop and yield losses. Plant tissue, particularly root tissue, is damaged by nematode feeding. Such feeding can cause mechanical tissue damage and the accompanying injection of nematode enzymes can cause further tissue disintegration. Nematode infections of roots result in root galls, and distortions in root growth. Similar symptoms accompany nematode infections of other parts of the plants. Important plant pathogenic nematodes include: root knot nematodes, Meloidogyne (e.g., Meloidogyne incognita); cyst nematodes Heterodera (e.g., H. glycines, H. schachtii) particularly for soybean and sugar beet; lesion nematodes Pratylenchus; burrowing nematodes Radopholus similis; stem and bulb nematodes Ditylenchus (e.g., D. dipsaci); citrus nematodes Tylenchulus; seed gall nematodes Anguina: and foliar nematodes Aphelenchoides.
Nematode infection can be accompanied by bacterial or fungal infection. In such plant-disease complexes, damage caused by nematodes can lead to enhanced severity of bacterial or fungal infection. For example, Fusarium and Verticillium wilts, Pythium damping off, Rhizoctonia and Phytophthora root rots as well as Pseudomonas solanacearum and Corynebacterium insidiosum bacterial wilts are enhanced by nematode infection. In addition, several nematodes (Xiphinema, Longidorus, and Trichodorus) are vectors for plant pathogenic viruses.
Plant pathogenic nematodes have been controlled by use of crop rotation or by the use of chemical fumigants and nematicides. In addition, a variety of nematode resistant plants have been developed using conventional breeding methods. Several methods of biocontrol have also been applied to plant pathogenic nematode control. A number of fungi, including Verticillium chlamydosporium and several bacteria, including Bacillus penetrans are reported to be effective against nematodes.
Miller and Sands (1977) J. Nematology 9:192-197 have reported that chitinase is toxic to certain nematodes. Chitin is a component of the cell wall of fungi, of nematode eggs and possibly present in nematode larvae which is degraded by chitinase and other chitinolytic enzymes. The antimycolytic activity of soil bacterial isolates including strains of Bacillus, Pseudomonas and Arthrobacter have been associated with chitinase production (Mitchell and Alexander (1961) Nature 190:109-110; Sneh (1981) Phytopath. Z. 100:251-256). To applicants' knowledge, there have been no reports associating the nematode-antagonistic activity of bacterial soil isolates to chitinase production.
Suslow et al. EPO patent application 157351, published Oct. 9, 1985 describe a method for control of chitinase-sensitive plant pathogens, including nematodes, in which a rhizobacterium root-colonizing strain, for example Pseudomonas fluorescens, genetically altered to contain a chitinase gene, is employed as a plant inoculum. Jaworski et al. EPO patent application 171381, published Feb. 12, 1986 describe a method of nematode control using soil bacteria in which the bacteria are transformed to contain glycosidase enzymes and/or chitinase and thus to inhibit nematodes. Jaworski et al. also report that two P. fluorescens strains (701E1 and 112-12) isolated from soybean or corn roots inhibited infection of soybean or tomato plants by root knot nematodes. The mechanism by which the P. fluorescens isolates inhibited nematodes was not described.
Strains of Pseudomonas cepacia have been reported to produce a variety of antimicrobial compounds active against bacteria and/or fungi. P. cepacia ATCC 17759 is reported (German Patent No. 3149608, issued 1983) to produce substituted tropolones that are effective against bacteria and Streptococci. P. cepacia ATCC 39356 is reported (U.S. Pat. No. 4503218, issued 1985; Parker et al. (1984) J. Antibiot. 37:431-440) to produce Cepacin A and B, described as substituted furanones, which are effective as bactericides. Japanese patents 54-157501 (1979) and 55-122794 (1980) report "novel antibiotics" produced by P. cepacia strains BN 229-A and BN 225, respectively, which are effective against bacteria. Japanese Patent 53-127894 (1978) reports that P. cepacia strain KB-1 produces altericidin A, B and C which are effective against Eumycetes and effective against the causative agents of black spot of pear, gummy stem blight and scab of cucumber. P. cepacia ATCC 39277 is reported (U.S. Pat. No. 4,601,904, issued 1986) to produce xylocandin A and B which are active against yeast and fungi. P. cepacia strain 4137 is reported (Smirnov et al. (1982) Antibiotiki (Mosc.) 27:577-580) to produce a yellow pigment fraction, possibly a derivative of hydroxyphenazine carboxylic acid, which is active against bacteria, yeast and fungi. Pyrrolnitrin and aminopyrrolnitrin, which have antifungal activity, have been reported by Elander et al. (1968) Appl. Microbiol. 16:753-758, to be produced by P. multivorans (now included in the species P. cepacia). Recently these compounds have been reported to be produced by Pseudomonas cepacia strains having broad-spectrum antifungal activity (Dart et al. U.S. patent application Ser. No. 106,986 and Lambert et al. (1987) Appl. Environ. Microbiol. 53:1866-1871). Applicants know of no reports that associate the antimicrobial compounds produced by P. cepacia with nematode inhibition.
In several instances, P. cepacia strains have been reported to be useful for plant protection from plant pathogenic fungi. Kawamoto and Lorbeer (1976) Plant Dis. Reptr. 60:189-191 report that inoculation with P. cepacia strain 64-22 protected onion seedlings from damping off caused by a strain of Fusarium oxysporum f.sp. cepae. This P. cepacia was reported to be recovered from the root, root stem and seed coat of seedlings grown from inoculated seeds. P. cepacia 64-22 was, however, confirmed to be an onion pathogen itself. Becker et al. (1984) in Proceedings of British Crop Protection Conference (Pests and Diseases), Volume I, Nov. 19-22, 1984, BCPC Publications, Croyden, U.K. pp. 365-370 report that the P. cepacia strain of Kawamoto and Lorbeer, 1976, which they designate strain A4, is inhibitory in vitro to growth of a variety of fungi, including Pythium ultimum, Rhizoctonia cerealis, Fusarium nivale and Ceratocystis ulmi among others. Cavileer and Peterson (1985) Abstract No. 522, Phytopathological Society Annual Meeting report that inoculation with a strain of P. cepacia protected China Aster against wilt caused by Fusarium oxysporum f. sp. callistephi in greenhouse and field tests. Lumsden (1982) Phytopathology 72:709 reports that a strain of P. cepacia is antagonistic to the fungus Pythium aphanidermatum and can protect cucumber seedlings from infection. Lumsden and Sasser U.S. Pat. No. 4,588,584, issued May 13, 1986, describe the protection of cucumber and pea from Pythium disease employing seed inoculation with a new biotype of P. cepacia designated SDL-POP-S-1. Birch (1986) Australian Plant Pathol. 15:51-56 reports that P. cepacia isolates from the sugarcane rhizosphere strongly inhibited in vitro growth of Pythium graminicola but did not reduce disease in plant pot trials. Knudsen and Spurr (1987) Plant Dis. 71:442-445 report that leaf inoculation by a strain of P. cepacia controlled peanut leaf spot (Cercospora arachidicola). Elad and Chet (1987) Phytopathology 77:190-195 report that six bacterial strains, including a strain of P. cepacia, which were isolated from the rhizosphere of plants infested with Pythium were effective biocontrol agents for that fungus. Disease control was obtained in cucumber, bean, pepper, melon, tomato and cotton. It is also reported therein that suppression of disease is significantly correlated with competition for nutrients between the bacteria and germinating oospores of the fungus. Jones and Roane (1982) Can. J. Microbiol. 28:205-210 report that a strain of P. cepacia inhibited growth and spore formation of Septoria nodorum.
Becker et al. (1985) Med. Fac. Landbouww. Rijkuniv. Gent. 50/3b report the isolation of a number of bacterial isolates from the phytosphere of maize and chicory which are inhibitory to fungal growth in vitro. Fungal-inhibitory isolates included P. cepacia, P. fluorescens, Erwinia serratia and Bacillus. The Pseudomonas isolates are reported to display in vitro activity against a broad spectrum of fungi.
Dart et al. U.S. Pat. No. 4,798,723 describe a class of P. cepacia rhizosphere isolates designated P. cepacia type Wisconsin that are non-phytopathogenic, have broad spectrum antifungal activity, are able to colonize leaves and roots of a variety of plants and are able to protect plants which they colonize from fungal disease. These P. cepacia strains, exemplified by P. cepacia strain 526, were found to be active in vitro against several strains of Fusarium, Sclerotinia sclerotiorum, Macrophomina phaseolina, Colletotrichum lindemuthianum and Rhizoctonia solani among others.
While two rhizosphere isolates of P. fluorescens have been reported to inhibit nematode invasion (Jaworski et al., 1986), to applicants' knowledge there have been no reports that P. cepacia root isolates are effective for plant protection from nematode invasion.